Analysis of sypK gene in Vibrio fischeri Biofilms

Who am I?

picture of lab

I’m Aishwarya Raj, a senior in high school

at the Illinois Math & Science Academy.

My research investigation was conducted

at University of Loyola: Stritch School of

Medicine.

BackgroundVibrio fischeri & Biofilms

Vibrio fischeri works with the squid (Euprymna scolopes) by

colonizing a symbiotic light organ inside the squid when the

squid is young (Mcfall-Ngai, 1996). The interesting nature of

the symbiotic relationship is that conditions within the light

organ are favorable only to V. fischeri and accordingly only

allow V.fischeri to colonize the light organ (Mcfall-Ngai, 1996).

Using this relationship, there have been various research

conducted on identifying the process and key components

through which a biofilm develops or does not develop.

Gram-negative, bioluminescent bacteria found in temperate

and subtropical waters.

sypK

Putative oligosaccharide translocase.

Promotes bacterial motility and biofilm formation.

Uncooperative relationship with bioluminescence as

increased sypK expression results in decreased

bioluminescence.

Purpose

The purpose of this investigation was to

analyze two phenotypes of the bacterium

Vibrio ficheri, motility and biofilm formation

through the gene sypK, and identify

whether these phenotypes can be

expressed independent of each other as

well as identify which amino acids are

essential for sypK to express both or either

phenotypes.

HypothesisIt was theorized that when testing to understand whether

Vibrio fischeri could exhibit one of its phenotypes

independent of the other, the bacterium would exhibit

motility rather than biofilm formation. Furthermore it was

also theorized that the sypK gene could be mutated to

find strains of V. fischeri in which only one phenotype was expressed. This is because the sypK gene codes for a

translocase protein whose function is simply to flip

polysachrides (biofilm) across the bacteria membrane so

essentially mutating the sypK gene would result in

inactivating biofilm formation. Furthermore it may be

possible that the sypK gene is needed for the bacterium

to transform from state of biofilm production to free floating state and consequently over expression of sypK

may increase motility while decreasing biofilm formation.

Methodology

I analyzed SypK function by conducting various

experiments in which I assessed the motility(movement) of strains of bacteria that express mutant versions of the sypK gene that are unable to promote biofilm formation. Three possible outcomes which were conceivable in this investigation were that sypK can have a biofilm and motility defect, biofilm defect but no motility defect, or no biofilm defect but only motility defect. I hoped to understand if all biofilm-defective sypK mutants also exhibit a defect in their ability to promote motility, or if the two phenotypes can be separated; if the latter is true, this might suggest that distinct amino acids are necessary for the two phenotypes associated with SypK.

In addition, I also conducted experiments in which I identified additional sypK mutants by taking mutagenized sypKplasmid and transferring it from an E. coli strain into V. fischeri and selecting for the presence of the plasmid by inoculating the culture onto petri plates with selective media in a process called conjugation. I then looked for qualitative observations such as smooth colonies (wrinkled is normal) or those that are delayed in forming biofilms. I collected strains with biofilm-defective phenotypes and verified the phenotype. Then, I analyzed if the biofilm-defective sypK mutants also exhibited defects in inducing motility. In order to check if there is a motility defect, equal concentrations of V. fischeri strains containing mutants were spotted onto motility plates and checked after 3 hours to see if the bacteria formed a ring around the place of origination, which occurs as the bacteria swim, to the same extent as occurs with the nonmutagenized control.

I was able to generate some mutant strains which exhibited heightened motility while still displaying a lack of biofilm formation. DNA samples of these mutants were then sent out to ATGC for gene sequencing and then compared to sypK wild type sequence to observe any viable mutations. IPTG experiments were also conducted to see if inducible mutant sypKstrains could bring back biofilm production.

ResultsBiofilm Assay 9/24/2014:

This biofilm assay is of the control strains(6469(negative control)

and 6470(positive control) and the known mutant strains for

observing baseline qualitative changes to biofilm over time.

Biofilm Assay 11/19/2014

This biofilm assay

portrays all strains of

V.fischeri observed

over time including

the mutant strains

investigated(AR1

(same as AR3) and

AR2) which do not

display the normal

wild type phenotype

of wrinkled biofilms.

MeaningThe graphs of motility in the previous slide depict

the comparison of the mutant strains (AR2 and AR3) in

comparison the control strains in terms of motility or

movement over time. From these graphs it is apparent

that the mutant strains exhibit above average motility as

AR3 especially is more motile than the positive

control(6470).

Qualitative observations

Hour 2 Hour 2 Hour 3 Hour 4

Displays how the various V. fischeri strains look during motility

experiments at various time intervals.

Conclusion

Evidence from investigation suggests that amino acids required

for biofilm formation are not the same amino acids required for

motility as the select V.fischeri strains were able to exhibit motility

without expressing biofilm formation.

Through generation of mutant strains it was possible to separately

express sypK phenotypes motility and biofilm formation.

X The gene sequence of the mutant strains held no significant

mutations in order to examine amino acid sequence.

Future targets & RelevanceAlthough there were no significant mutations in

the mutant strains, the biofilm phenotype was not properly expressed. Consequently it was theorized that the amount of sypK expression is altered, which could increase motility while decreasing biofilm formation. In the future this hypothesis can be tested via strains in which sypK expression is controlled by an exogenous inducer, IPTG, and determining if altered sypK levels cause a defect in biofilm formation.

The information from this can then be used in conjunction with other biofilm producing bacteria to see if biofilm formation can similarly be switched on or off. If successful, this would mean that for a large portion of antibiotic resistant bacteria, the biofilm production can simply be signaled to decrease whilst then delivering antibiotics for proper effects.

To elaborate on the idea

presented in the previous

slide, it was theorized that

the level of sypK expression

may determine whether

biofilm production and

bacterial adherence is

promoted or whether motility

is promoted. As a result when

sypK was overexpressed it

may be possible that while

motility was promoted,

biofilm production was down

regulated. This would then

explain why the mutant

strains exhibited above

average motility.

ReferencesBrennan, C. A., Mandel, M. J., Gyllborg, M. C., Thomasgard, K. A., & Ruby, E. G. (2013).

Genetic determinants of swimming motility in the squid light-organ symbiont Vibrio fischeri.

MicrobiologyOpen, 576-594. http://dx.doi.org/10.1002

Mcfall-Ngai, M. (1996). Studies of the Symbiotic Relationship Between Animals and Microorganisms.

Science Spectra, (4), 14–19.

Miyashiro, T., Oehlert, D., Ray, V. A., Visick, K. L., & Ruby, E. G. (2014). The putative oligosaccharide

translocase SypK connects biofilm formation with quorum signaling in Vibrio fischeri.

MicrobiologyOpen, 1–13.

Morris, A. R., & Visick, K. L. (2013). The response regulator SypE controls biofilm formation and colonization

through phosphorylation of the syp-encoded regulator SypA in Vibrio fischeri. Molecular

Microbiology, 87(3), 509–525.

Shibata, S., Yip, E. S., Quirke, K. P., Ondrey, J. M., & Visick, K. L. (2012). Roles of the structural symbiosis

polysaccharide (syp) genes in host colonization, biofilm formation, and polysaccharide

biosynthesis in Vibrio fischeri. Journal of Bacteriology, 194(24), 6736–6747.

Visick, K. L. (2009). An intricate network of regulators controls biofilm formation and colonization by Vibrio

fischeri: MicroReview. Molecular Microbiology.

Contributions

My most sincere gratitude goes to my

parents, the student investigation research

staff at my school who guided me

throughout the process, University of Loyola:

Stritch School of medicine for allowing me

to conduct a research project, and my

advisors, Dr. Karen Visick and Ms. Cecilia

Thompson.